Soy isoflavones possess estrogenic properties, and reproductive tract tissues (including Leydig cells) express estrogen receptors (ER1 and ER2). Leydig cells are the predominant source of the male sex steroid hormone testosterone. Approximately 15% of infants fed soy-based formulas in the United States each year are exposed to the phytoestrogens genistein and daidzen, which are the predominant isoflavones in soybeans. Several reports in the literature have shown that exposure of rodents and non human primates to soy isoflavones affect testicular androgen secretion. However, the mechanisms by which soy isoflavones act in Leydig cells have not been investigated. The P.I. hypothesizes that disparities in serum androgen levels and Leydig cell steroidogenic capacity are linked to differences in the numbers of Leydig cells. Consistent with this hypothesis are recent observations indicating that genistein regulates Leydig cell mitosis. Also, soy isoflavones are known to regulate development of somatic cells in the testis, e.g. Sertoli cells. Therefore, cell-cell interactions, e.g., between Sertoli cells and Leydig cells, are subject to regulation by soy isoflavones. To address these issues, experiments will be performed to achieve the following aims: I. Soy isoflavone regulation of mesenchymal Leydig cell precursors;II. Mechanisms of genistein action in Leydig cells;and, III. Sertoli cell-mediated regulation of Leydig cells. The proposed study is relevant to public health and will be the first to define the mechanisms and molecular pathways of estrogen action in Leydig cells. Also, results will promote informed use of soy isoflavones in the diet, especially in neonates, in which the developing reproductive tract is sensitive to changes in the hormonal milieu. Furthermore, the present application will provide research training to undergraduate, professional and graduate students. The growing public concern for agri-terrorism puts veterinarians in the fore-front of research to aid defense of food supplies just as biomedical research is increasingly benefiting from investigation conducted in food animal species, e.g., use of pigs to study obesity and diabetes and dogs for cardiopulmonary diseases. Therefore, research training afforded by the present application will contribute to efforts geared to preparing future veterinarians to meet these challenges. PUBLIC HEALTH RELEVANCE: Soy isoflavones possess estrogenic properties, and reproductive tract tissues (including Leydig cells) express ER1 and ER2. Because proliferative activity in Leydig cells, which determines the final number of Leydig cells, is restricted to the prepubertal period, modulation of Leydig cell mitosis in the prepubertal period has consequences for Leydig cell numbers in the adult testis. The present application is designed to identify the mechanisms by which soy isoflavones cause long-lasting effects on testicular androgen secretion using the rat model. Experiments will be performed to address three Specific Aims: Soy isoflavones affect mesenchymal Leydig cell precursors in the prenatal and/or neonatal period, which alters Leydig cell proliferation and population (Aim I);Estrogen regulation of Leydig cell mitosis is mediated by interaction of signaling pathways, which involves the cell survival factor protein Akt (protein kinase B) acting in concert with estrogen receptor ER1, epidermal growth factor receptors (EGFR), insulin growth factor-1 receptors (IGF-1R) and mitogen activated protein kinases (MAPK) (Aim II);and, Action of soy isoflavones in Sertoli cells affects Leydig cells (Aim III). Timed pregnant dams will be fed diets containing low and high isoflavone levels (5, 1000 ppm) in the perinatal period. Male offspring will be analyzed for Leydig cell proliferation in prepubertal and pubertal rats. In addition, the number of Leydig cells will be enumerated by stereology in the adult testis at 90 days of age. Analysis of genistein action regulating Leydig cell mitosis will involve use of pharmacological inhibition and gene silencing techniques to investigate the role of Akt, ER1, EGFR and IGF1-R. The possibility that soy isoflavones act directly in the seminiferous epithelium will be investigated by analysis of Sertoli cell differentiation and maturation. In addition, Sertoli cell-Leydig cell interactions will be evaluated by culture of Leydig cells in Sertoli cell-conditioned media. The proposed experiments will be the first to investigate the mechanisms of soy isoflavone action in Leydig cells. In particular, the EDS- and busulfan-treated rat models provide opportunities to study cell proliferation and cell-cell interactions in vivo. These models have contributed immensely to our understanding of testicular biology but have not been used in toxicological studies. Also, the hypothesis that estrogenic compounds, and perhaps other endocrine-active agents, may exert indirect effects in Leydig cells by action exerted in Sertoli cells has not been explored. Together, these approaches will yield information not previously described for phytoestrogen action in testicular cells. The information will contribute to ongoing about potential reproductive toxicity of soy-based diets especially in neonates. The growing public concern for agri-terrorism represents a challenge to veterinarians who have responsibilities for defense of food supplies. Also, biomedical research is increasingly benefiting from investigation conducted in food animal species, e.g., use of pigs to study obesity and diabetes and dogs for cardiopulmonary diseases. Research training to be afforded by the present application to professional veterinary students will contribute to efforts geared to preparing future veterinarians to meet these challenges.